Bovine herpesvirus type 1 (BHV-1) causes infectious bovine rhinotracheitis, a highly contagious disease of cattle, characterized by severe inflammation of the upper respiratory passages and trachea. This virus is also an etiologic agent of vulvovaginitis, balanoposthitis, abortion, conjunctivitis, meningoencephalitis, and fatal systemic infections. Lupton and Reed, Evaluation of Experimental Subunit Vaccines for Infectious Bovine Rhinotracheitis, Am. J. Vet. Res. 41, 383-90 (1980). Natural infections have been described in cattle, goats, swine, water buffalo, wildebeests, and ferrets. Experimental infections have been established in mule deer, goats, swine, ferrets and rabbits. Fulton et al., Prevalence of Bovine Herpes Virus-1, Bovine Viral Diarrhea, Parainfluenza-3, Bovine Adenoviruses-3 and -7, and Goat Respiratory Syncythial Viral Antibody in Goats, Am. J. Vet. Res. 43, 1454-57 (1982). Infected cattle may have temperatures ranging from 104.degree. to 107.degree. F., a heavy mucous nasal discharge, depression, loss of appetite, increased rate of respiration, labored breathing, and coughing. Superinfection by Pasturella haemolytica often results in severe pneumonia.
The severity of the illness resulting from BHV-1 infection depends upon the virus strain and on the age of the animal affected. Encephalitis and death are generally restricted to calves. After recovery from BHV-1 infection, animals may show clinical signs of recurrent disease without being reexposed to the virus. Recurrent disease without reexposure occurs because the virus remains dormant in the neurons of the sensory ganglia of its host and can be reactivated even after long periods. Dexamethasone treatment can provoke release of the virus from the neurons and viral shedding with or without clinical signs and symptoms of active BHV-1 disease. Ackermann, et. al., DNA of Bovine Herpes Virus Type 1 in the Trigeminal Ganglia of Latently-Infected Calves, Am. J. Vet. Med., 43, 36-40 (1982). Additionally, stress caused by transportation or parturition often causes reactivation of latent BHV-1 in infected calves.
Infections caused by BHV-1 were first recognized in the United States in the mid-1950's. These infections had, however, existed in Europe for at least one hundred years and had been described scientifically in 1928. By the mid-1960's, respiratory disease caused by BHV-1 was causing losses in the United States estimated at $25 million. Additional losses associated with BHV-1 infections resulted from abortion storms, dramatic losses in milk yield, fatal metritis, enteritis, and meningitis. Since 1972, more severe forms of BHV-1 respiratory infections have become widespread in Western Europe and Canada. The increased infection severity suggests emergence of more virulent BHV-1 strains. New infectious bovine rhinotracheitis outbreaks probably result from exposure to imported asymptomatic BHV-1 carriers or exposure to infected animals prior to onset of clinical disease.
Currently, three types of infectious bovine rhinotracheitis vaccines are available: inactivated adjuvant vaccines, subunit vaccines, and modified live virus (MLV) vaccines. Inactivated BHV-1 vaccines are produced by treating the virus with formalin or ethanol and heat or by ultraviolet irradiation. Subunit BHV-1 vaccines are prepared by solublizing BHV-1 infected cell cultures with nonionic detergents. The early modified live virus vaccines were designed for parenteral administration and consisted of virus attenuated by rapid passage in bovine cell cultures. Schwartz, et al., Modification of Infectious Bovine Rhinotracheitis (IBR) Virus in Tissue Culture and Development of a Vaccine, Proc. Soc. Exp. Biol. Med. 96,453-58 (1957). Subsequent parenterally administered MLV vaccines were attenuated by adaptation of the virus to porcine or canine cell cultures, adaptation to growth and cell culture at 30.degree. C., or by selection of heat-stable mutants (56.degree. C. for 40 minutes). The presently available MLV vaccines are administered intranasally and are attenuated by serial passage in rabbit cell cultures or by treatment of the virus with nitrous acid followed by selection of temperature-sensitive mutants, that is, viruses that replicate efficiently at about 33.degree.-37.degree. C., but not at 39.degree. C. Vaccine Preparation Techniques, Ed. by J. I. Duffy, Noyes Data Corporation, Parkridge, N.J. 1980, pp. 98-102; Lloyd-Evans, L. P., "Tracherine." Infectious bovine rhinotracheitis vaccine. Temperature-specific IBR virus strain RLB 106 ts. produced by the Technical Services Department, Smith Kline Animal Health Ltd., Welwyn Garden City, Hertfordshire, United Kingdom, 1979, pp. 1-78.
Each of the available types of infectious bovine rhinotracheitis vaccines has, however, proved unsatisfactory in commercial use. Inactivated adjuvant vaccines are undependable and inadequately protective and generally inferior to MVL vaccines. For example, an inactivated virus vaccine with a 5-component adjuvant was found entirely ineffective in producing immunity, preventing disease, and suppressing propagation and reexcretion of virulent virus. Msolla, et. al., Vaccination Against Infectious Bovine Rhinotracheitis, Vet. Record 104, 535-36 (1979). Following challenge exposure to virulent virus, vaccinated animals exhibited clinical signs and virus excretion responses virtually identical to unvaccinated animals and, in marked contrast to calves recovered from natural infection, transmitted virulent virus to in-contact controls. The lack of efficacy of inactivated virus vaccines has also been demonstrated by inducing tracheitis, tachypnea, pyrexia, occular and nasal discharges, and severe ulceration of the nasal mucosa by exposing animals previously vaccinated with an inactivated polyvalent vaccine to BHV-1 virus.
Extensive utilization of modified live virus vaccines has reduced the frequency of infectious bovine rhinotracheitis. However, none of the available modified live virus vaccines is entirely satisfactory for use in infectious bovine rhinotracheitis control programs. For example, bovine kidney passaged Colorado Strain BHV-1 modified live virus vaccine was administered to calves intravenously, intrapreputially, or intranasally. Sheffy and Rodman, Activation of Latent Infectious Bovine Rhinotracheitis, J. Am. Vet. Med. Assoc. 163, 850-51, (1973). Beginning ten weeks after vaccination, dexamthasone was administered to some of the calves to induce release of latent virus. In dexamethasone treated calves, BHV-1 was found in the respiratory and genital tract tissues of intrapreputially and intravenously vaccinated calves and the respiratory tract tissues of intranasally vaccinated calves. BHV-1 virus was not found in unvaccinated calves. These data, therefore, clearly demonstrate that latent BHV-1 infections resulted from innoculation with the modified live virus vaccine. Because stress can also cause release of latent virus, prevention of latent infections is important to avoid spread of pathogenic virus and disease to non-vaccinated cattle.
Vaccination with available modified live virus preparations is also ineffective in preventing latent infection following exposure to virulent BHV-1. To demonstrate this inefficacy, a modified live virus vaccine, obtained by passing BHV-1 forty-three times in porcine testes cells followed by eight passages in monolayer cultures of bovine testes at 30.degree. C. was employed. Narita, et al., Neural Changes in Vaccinated Calves Challenge Exposed With Virulent Infectious Bovine Rhinotracheitis Virus, Am. J. Vet. Res. 41, 1995-99 (1980). Following vaccination, the calves were challenge exposed to virulent BHV-1 (Los Angeles Strain). Latent viral infection was demonstrated by administering dexamethasone forty-nine days following the challenge exposure and observing signs of recurrent infection. Calves vaccinated with a temperature-sensitive intranasal vaccine were also incompletely protected against latent viral infection following exposure to virulent virus. Rossi and Kiesel, Effect of Infectious Bovine Rhinotracheitis Virus on Virus Shedding in Challenge Exposed Calves Treated With Dexamethasone, Am. J. Vet. Res. 43, 1576-79 (1982).
In addition to the demonstrated inefficacy in preventing latent viral infections, available modified live virus vaccines often fail to prevent active disease. Cattle have developed respiratory tract disease and conjunctivitis following modified live virus vaccination. Also, periodic shedding of BHV-1 and development of mild clinical symptoms have been described after intranasal vaccination of cattle with modified live virus vaccine. Actively infected cattle and those shedding virus create a danger of disease transmission when these cattle come into contact with non-vaccinated animals.
The presently invented modified live virus vaccine, utilizing a BHV-1 mutant that is thymidine kinase negative (TK.sup.-) and temperature resistant (tr), overcomes many of the problems that have limited the use of currently available vaccines. This mutant lacks the ability to induce thymidine kinase activity in cells it infects and it can replicate efficiently in cells at 39.degree. C. These characteristics directly contribute to its superiority as a vaccine. Virus encoded thymidine kinase activity is important for BHV-1 virulence, for replication in nerve cells which lack inherent thymidine kinase activity, and for recrudescense. This mutant provides a stronger immunologic response than temperature sensitive mutants because its resistance to elevated temperatures allows it to replicate efficiently in tissues deep within the body and in febrile animals. Intranasal administration of the TK.sup.- tr BHV-1 mutant in calves does not result in symptoms of active infectious bovine rhinotracheitis disease. Further, it is now well-established that TK.sup.- herpes viruses mutliply poorly in nerve cells and are less likely to cause latent infection. It is also difficult to reactivate TK.sup.- tr herpes viruses and they do not recrudesce. Yet, vaccines utilizing these mutants are highly effective in preventing infection by virulent BHV-1. Additionally, vaccination with these mutants protects against latent infection development following exposure to virulent virus. Field & Wildy, The Pathogenicity of Thymidine Kinase-Deficient Mutants of Herpes Simplex Virus in Mice, J. Hygiene (Cambridge) 81-267-77 (1978); Field & Darby, Pathogenicity in Mice of Strains of Herpes Simplex Virus Which Are Resistant To Acyclovir In Vitro and In Vivo, antimicrobial agents and chemotherapy 17, 209-16 (1980); Tenser, et al., Trigeminal Ganglion Infection by Thymidine Kinase-Negative Herpes Simplex Virus, Science 205, 915-17 (1979).